Week 3 HW: Cell-Free Protocols

Week 3 — Cell-Free Reaction Design

3. Assignment: CFPS Reaction Design for sfGFP, PETase, and Reteplase

The objective of this assignment was to design three cell-free protein synthesis (CFPS) reaction compositions, one for each target protein: sfGFP, PETase, and Reteplase.

The main challenge was not simply to maximize protein production, but to reduce the cost of producing functional protein. For this reason, I compared different energy systems, considered the folding requirements of each protein, and identified reagents that could potentially be reduced without significantly affecting protein activity.

I used the CFPS Coopetition platform to explore the reference formulations and submit my experimental designs.

A. Compare Cell-Free Reaction Compositions

A.1. Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?

Potassium glutamate and magnesium glutamate are both important for protein synthesis, but they have different functions.

Potassium glutamate helps maintain an ionic environment similar to the bacterial cytoplasm, supporting the activity of the transcription and translation machinery. Magnesium ions, on the other hand, are directly involved in ribosome stability, RNA structure, and interactions with nucleotides.

Their optimal concentrations can change depending on the lysate, energy system, and other reagents present in the reaction.

Magnesium glutamate is generally more sensitive. A small change in magnesium concentration can significantly affect protein synthesis because magnesium interacts with ATP, RNA, and other negatively charged molecules. Potassium glutamate usually has a broader range of acceptable concentrations.

Therefore, magnesium concentration must be carefully considered when modifying a CFPS formulation.

A.2. Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.

The main difference between these formulations is how they supply energy and nucleotides for transcription and translation.

PEP+NTP formulations directly provide nucleotide triphosphates (ATP, CTP, GTP, and UTP), while phosphoenolpyruvate (PEP) supports ATP regeneration. This system provides readily available energy and nucleotide substrates, but several of its components can be relatively expensive.

Ribose+NMP formulations instead use ribose as a metabolic substrate and nucleotide monophosphates (AMP, CMP, GMP, and UMP). These components are generally less expensive, but the reaction depends more heavily on the metabolic enzymes remaining in the lysate to regenerate the nucleotides required for transcription.

The trade-off is therefore between reagent cost and metabolic dependence.

For this homework, this distinction was especially important because the reference data showed that different proteins responded differently to the two energy systems.

The PETase and Reteplase values represent assay product measurements associated with enzymatic activity, not protein concentration or titer.

The Ribose formulation appeared relatively promising for sfGFP and PETase, while the difference was much greater for Reteplase. This influenced my decision to retain the PEP energy system for the Reteplase design.

A.3. Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.

I compared the traditional PANOx-SP formulation described by Jewett and Swartz (2004) with the more recent cost-optimization approach discussed by Olsen et al. (2026).

PANOx-SP uses phosphoenolpyruvate as an energy source and supplies nucleotide triphosphates directly. It also includes salts, amino acids, and supporting components such as NAD, CoA, spermidine, putrescine, and oxalate. These components help create an environment that supports sustained transcription and translation.

In contrast, cost-oriented CFPS formulations investigate whether reagent concentrations can be reduced or alternative energy and nucleotide systems can be used while maintaining sufficient protein production.

The important difference is not that one formulation is universally better than the other. Instead, they represent different optimization priorities.

This comparison helped me understand why removing a reagent is not automatically an improvement. A cheaper reaction may still be less cost-effective if functional protein production decreases too much.

B. Design One Reaction Composition for Each of the Three Proteins

B.1. Experimental design strategy

I submitted three experimental CFPS compositions through the Coopetition platform.

Each reaction has a total volume of 20 µL (20,000 nL), including:

• 5,000 nL of lysate
• 2,000 nL of DNA template
• 2,000 nL of base buffer
• 11,000 nL of additional reagents and nuclease-free water

All reagent volumes were selected in increments of 25 nL.

Instead of using the same strategy for every protein, I adapted each composition to the protein’s folding requirements and the reference data.

Composition 1 — sfGFP: Simplified Ribose+NMP

Sample ID: david_toledo_sfgfp_minimal_ribose_nmp_v1

Target: Superfolder green fluorescent protein (sfGFP)

Design objective: Reduce the cost of a functional sfGFP reaction by simplifying a Ribose+NMP formulation.

sfGFP is a relatively robust protein that does not require disulfide-bond formation. Because of this, I considered it a suitable target for testing a simplified reaction composition.

I selected a Ribose+NMP energy system and reduced several supplementary components compared with the reference formulation. The idea was to take advantage of the metabolic capacity of the lysate while avoiding reagents that might contribute relatively little to functional protein yield.

Submitted composition:

Hypothesis

A simplified Ribose+NMP formulation may reduce the cost of functional sfGFP production while maintaining sufficient protein yield. Even if the final sfGFP concentration is lower than that of the reference, the formulation could still be more cost-effective if the reduction in reagent cost is proportionally greater.

Expected outcome

I expect this formulation to provide a useful test of how much the reaction can be simplified before functional sfGFP production becomes too low.

Composition 2 — PETase: Reduced DsbC in a Ribose+NMP Formulation

Sample ID: david_toledo_petase_low_dsbc_ribose_v1

Target: PETase (TfCut2)

Design objective: Reduce the cost of folding assistance while maintaining PETase enzymatic activity.

Unlike sfGFP, PETase contains one disulfide bond. Therefore, correct folding is more important when evaluating its activity.

One reagent that attracted my attention was DsbC, a disulfide-bond isomerase that helps correct incorrectly paired disulfide bonds. DsbC is also particularly expensive in the available reagent inventory.

I decided to test whether PETase could maintain sufficient activity with a lower DsbC concentration.

Submitted composition:

Main modification: DsbC was reduced from 5 µM to 1 µM.

Hypothesis

Reducing DsbC concentration from 5 µM to 1 µM may substantially lower the reaction cost without causing an equally large decrease in PETase activity.

Since PETase contains only one disulfide bond, it may require less folding assistance than a more structurally complex protein such as Reteplase.

Expected outcome

The experiment will help determine whether lower DsbC concentrations are sufficient to produce functional PETase and whether the cost reduction compensates for any loss of activity.

Composition 3 — Reteplase: PEP+NTP with Reduced DsbC

Sample ID: david_toledo_reteplase_pep_low_dsbc_v1

Target: Reteplase

Design objective: Reduce the cost of DsbC while preserving an energy and redox environment suitable for oxidative protein folding.

Reteplase was the most challenging target because it contains nine disulfide bonds that must form correctly for the protein to be functional.

The reference results also showed a large difference between the two energy systems: the PEP formulation produced a much stronger activity-associated signal than the Ribose formulation.

For this reason, I decided not to replace the PEP+NTP system. Instead, I focused on a more controlled modification: reducing DsbC while preserving the other components of the reference formulation.

Submitted composition:

Main modification: DsbC was reduced from 5 µM to 3 µM.

I preserved the GSSG/GSH redox system because Reteplase requires an oxidizing environment to form its disulfide bonds.

Hypothesis

Reducing DsbC from 5 µM to 3 µM may improve the cost per unit of functional Reteplase activity while maintaining enough disulfide isomerase activity for correct protein folding.

Expected outcome

I expect this experiment to reveal whether a moderate reduction in DsbC is compatible with functional Reteplase production. Because this protein has nine disulfide bonds, a loss of activity is possible, and the cost-effectiveness of the formulation must be determined experimentally.

B.2. Comparison of the three designs

The three formulations follow the same general goal but address different biochemical challenges.

For sfGFP, I prioritized simplifying the reaction. For PETase, I explored whether folding assistance could be reduced. For Reteplase, I preserved the reference energy and redox systems while making a smaller adjustment to an expensive reagent.

These designs have been submitted, but their performance has not yet been experimentally measured.

B.3. Community Reagents

I reviewed the reagent proposals submitted by other participants and voted for FkpA (E. coli chaperone), proposed by Xiangtao Shi.

I selected FkpA because it may help reduce protein aggregation and assist folding, potentially complementing DsbC.

This is particularly relevant to Reteplase, where producing the protein is not sufficient; the protein must also fold into an active structure.

The proposal also referenced research showing that FkpA and DsbC can work together to improve folding and assembly of complex proteins in cell-free systems.

I considered this a useful approach because it addresses a different part of the folding problem. DsbC helps rearrange incorrectly formed disulfide bonds, while FkpA can provide additional folding assistance.

My vote was successfully registered on the Community Reagents platform.

B.4. CFPS Coopetition Forum

Forum: cfps-coopetition

Still working on it jejej

C. Asimov Kernel — Optional

haven’t done this yet T_T

D. Discussion and Conclusions

This assignment helped me understand that CFPS optimization depends on more than simply selecting the cheapest available reagents.

The best formulation depends on the protein being produced, the metabolic capacity of the lysate, the energy system, and the requirements for correct folding.

The reference results suggested that Ribose+NMP systems may be worth exploring for proteins such as sfGFP and PETase, while Reteplase benefits substantially from the PEP-based formulation.

Another important consideration was the cost of DsbC. Because this reagent is expensive, reducing its concentration could potentially improve reaction economics. However, its contribution to functional protein production must be evaluated experimentally, particularly for Reteplase.

Overall, my three submitted compositions explore the balance between reagent cost and protein functionality using different approaches.

The next step will be to compare their experimental performance against the reference formulations and determine whether the proposed changes actually improve the cost per unit of functional protein.

E. Use of AI

I used ChatGPT (GPT-6, OpenAI) as a research assistant during the design process.

The tool helped me compare CFPS energy systems, understand the functions of individual reagents, calculate reaction volumes and final concentrations, and develop hypotheses for the three experimental compositions.

Examples of prompts used during the process include:

• “Compare PEP+NTP and Ribose+NMP systems for cell-free protein synthesis.”
• “Help me design a low-cost sfGFP formulation using Ribose and nucleotide monophosphates.”
• “Could reducing DsbC from 5 µM to 1 µM improve the cost-effectiveness of PETase production?”
• “How can I reduce DsbC in a Reteplase reaction while preserving the conditions required for disulfide-bond formation?”
• “Check that the reagent volumes add up to exactly 20,000 nL.”

The proposed compositions and scientific reasoning were reviewed before submission. Experimental performance remains to be validated.

F. References

1. Jewett, M. C., & Swartz, J. R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and Bioengineering, 86(1), 19–26. https://doi.org/10.1002/bit.20026

2. Olsen et al. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications. https://doi.org/10.1038/s41467-026-69605-8

3. Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Cell-free gene expression: an expanded repertoire of applications. Nature Reviews Genetics, 21, 151–170. https://doi.org/10.1038/s41576-019-0186-3

4. Rasor, B. J., et al. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology, 69, 136–144. https://doi.org/10.1016/j.copbio.2020.12.012

5. CFPS Coopetition. https://coopetition.htgaa.org/

6. OpenAI